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This manuscript reviews carcinomas of the upper aerodigestive tract associated with genetic rearrangements of the nuclear protein of the testis (NUT) gene, also known as NUT midline carcinomas. The literature is reviewed regarding all reported cases of NUT midline carcinomas and the clinicopathologic features are discussed. Our current understanding of the molecular pathogenesis of the disease is also discussed, along with the differential diagnosis for undifferentiated or poorly differentiated malignancy of the upper aerodigestive tract.

Epithelial malignancies with characteristic chromosomal translocations and gene fusions are uncommon, but they are not as uncommon as once thought.1 Papillary thyroid cancers, renal cell carcinomas, and mucoepidermoid carcinomas of the salivary glands are among the growing number of epithelial malignancies to have been shown to be associated with chromosomal translocations. Recently, researchers have shown that nearly 50% of prostatic adenocarcinomas have fusions of the TMPRSS2 and ERG genes.2

Most of the upper aerodigestive epithelial malignancies are squamous cell carcinomas, either conventional type or variants, and the majority of these are related to the use of tobacco products and alcohol.3 Viruses also play a role in the malignancies, however, including human papillomavirus (HPV) and Epstein-Barr virus (EBV). Recently, a small percentage of upper aerodigestive malignancies have been shown to be associated with translocations involving the nuclear protein of the testis (NUT) gene located at 15q14 [nuclear protein of the testis midline carcinomas (NMCs)].4,5 This manuscript reviews NMCs, with particular attention to those of the upper aerodigestive tract.

BACKGROUND

In the year 1991, 2 cases of carcinoma were described involving the thorax that were shown to have t(15;19).6,7 Both involved the mediastinum and were considered thymic in origin. Since then, 22 total cases have been described (Table 1).4–17 Thirteen of these cases have developed in girls or women. The tumors have been noted to occur over a vast age range (3 to 78 y) and a little more than half the cases have been reported in patients over the age of 20 years. Ten cases have involved the head and neck and, of these, 9 have involved the upper aerodigestive tract. Ten other cases have been noted within the thorax, usually felt to be primary to the mediastinum/thymus (n=6). One reported tumor was thought to be primary to the bladder and 1 to the axial skeleton (diagnosed originally as a primitive neuroectodermal tumor).4,15

Originally, the tumors were believed to primarily affect children and young adults, perhaps reflecting the increased likelihood that tumors from these patients are analyzed for karyotype. In a recent series of primarily undifferentiated carcinomas from the upper aerodigestive tract, 4 of the 5 patients found to have translocations by fluorescent in situ hybridization (FISH) were older than any previously reported patients, including a 78-year-old woman with an undifferentiated carcinoma of the larynx.5 This is particularly interesting given the fact that many of the cases tested came from younger patients (age range 15 to 88 y).

CYTOGENETICS/GENETICS

The most common translocation involving the NUT gene is the t(15;19) (q13;p13.1).10 This translocation fuses the NUT gene on chromosome 15 to the BRD4 gene. In about one-third of cases, however, the NUT gene is fused to a different partner gene (NUT-variant midline carcinoma). Rarely, the NUT gene is fused to the BRD3 gene at 9q34.2.13 BRD3 and BRD4 are included in the human BET protein family, defined as possessing 2 chromatin-binding bromodomains, and an extraterminal domain of unknown function. These proteins are known to bind transcriptionally active chromatin.10 The function of the NUT protein, which is expressed only in brain and germ cells, is not known.10 It is believed that the bromodomain portions of BRD4 or BRD3 retain the fusion protein in the nucleus, bound to chromatin. Some recent data indicate that the fusion proteins block epithelial maturation and squamous differentiation. Indeed, siRNA-induced withdrawal of the fusion proteins leads to squamous differentiation and cell cycle arrest.13

CLINICAL, GROSS, AND HISTOLOGIC FEATURES

Patients with NMCs frequently present with mass-related symptoms (including superior vena cava syndrome when the tumors present in the mediastinum) from their primary tumor, and many have metastases at the time of diagnosis (Fig. 1).7,9,14–17 Nonspecific symptoms such as fever and weight loss have also been noted.

As most tumors have presented at advanced stage (lymph node metastases, bone metastases, and pleural carcinomatosis), tumors have infrequently been resected.4,10,17 Thus, gross descriptions are lacking. Of the 3 gross specimens we have seen at the University of Virginia, 2 were sinonasal and received piecemeal and showed grossly obvious hemorrhage and necrosis. One lesion was present in a laryngectomy specimen and was described as a 3 cm variegated, somewhat circumscribed mass. Nodal metastases were characterized by enlarged nodes with a necrotic, nonspecific appearance.

Histologically, tumors are usually composed of sheets of undifferentiated cells (Fig. 2).3,4,10,17 Occasional cases may have more nested malignant cells betwixt desmoplastic stroma (Fig. 3). Large areas of coagulative necrosis may be present. The cells have scant amphophilic or eosinophilic cytoplasm. Nuclei have irregular contours although they tend to be somewhat uniform in size. They have fine to vesicular chromatin and prominent nucleoli. Mitotic figures and apoptotic bodies are common. Squamous differentiation is frequently seen and does not necessarily predict the partner gene, as once believed.10 The differentiation may be subtle with slight streaming of the malignant cells or it can be very distinct, with maturing squamous cells and extracellular keratin formation. Some authors, including ourselves, have noted that the keratinization may seem abrupt, sometimes reminiscent of Hassel corpuscles, with sheets of immature cells juxtaposed to well-differentiated, mature squamous nests (Fig. 4).5 The well-differentiated areas may even become cystic.

ANCILLARY STUDIES

NMCs are epithelial and react with antibodies to keratins (although the staining may be focal).3,4,10 Most cases, when tested, have also been immunoreactive with antibodies to p63, even in histologically undifferentiated areas, consistent with squamous differentiation (Fig. 5). The majority of tumors are reportedly immunoreactive with antibody to CK7 and focal immunoreactivity with antibody to CK20 has sometimes been seen.4 CD34 immunoreactivity was seen in a little more than half the cases studied in 1 review, a unique finding for an epithelial malignancy.4 Immunoreactivity with antigens expressed in small blue cell tumors of childhood has not been noted. NMCs have reportedly been nonreactive with antibodies to desmin, myoglobin, smooth muscle actin, muscle actin, chromogranin, synaptophysin, leukocyte common antigen, placental alkaline phosphatase, S100 protein, α-fetoprotein, neuron-specific enolase, CD57, CD99, and HMB45. Evidence of EBV and HPV infection has never been identified in NMC using a variety of techniques, including RNA in situ hybridization, immunohistochemistry, and polymerase chain reaction (PCR) (personal observations).10

FISH, demonstrating rearrangement of the NUT gene, is currently required for the diagnosis of NMC (Fig. 6). Probes have been developed for the regions flanking the typical break point of the NUT gene on chromosome 15 and for the typical breakpoints with BRD4 and BRD3, however, they are not commercially available. Real-time–PCR primers have also been developed for the BRD4-NUT fusion oncogene, however, they are not frequently used and cannot be used to diagnose NUT-variant midline carcinomas.

To aid in the detection of NMC in laboratories that do not have the means to perform FISH or real-time–PCR, both polyclonal and monoclonal antibodies have been developed to the NUT protein.5 Because of the restricted expression of NUT, aberrant expression of NUT in the nuclei of nongerm cell tumors is theoretically diagnostic of NMCs. Using 70% nuclear staining as a cutoff, the polyclonal antibody to NUT showed 60% sensitivity and 96% specificity in our series of undifferentiated carcinomas of the upper aerodigestive tract. The new monoclonal antibody is more effective, with a sensitivity of 87% and specificity of 100% (data not yet published) (Fig. 7).

DIFFERENTIAL DIAGNOSIS

The differential diagnosis for NMCs of the upper aerodigestive tract includes other undifferentiated malignancies including undifferentiated carcinomas and poorly differentiated squamous cell carcinomas. The differential diagnosis is also dependent on the site of the malignancy. Within the upper aerodigestive tract, the differential diagnosis includes pediatric small blue cell tumors (eg, primitive neuroectodermal tumor, rhabdomyosarcoma, desmoplastic small round cell tumor, etc), melanoma, olfactory neuroblastoma, high-grade hematologic malignancies, endocrine carcinomas, and other undifferentiated or poorly differentiated carcinomas including sinonasal undifferentiated carcinomas and EBV-driven nonkeratinizing squamous cell carcinomas that occur most frequently in the nasopharynx (Table 2). Within the mediastinum and lung, the differential diagnosis is similar, with the exception of a few malignancies specific to the upper aerodigestive tract (eg, olfactory neuroblastoma) or thorax (eg, poorly differentiated thymic carcinoma). Germ cell tumors are also generally considered higher on one's differential diagnosis for mediastinal tumors than they are with upper aerodigestive tract tumors, especially as upper aerodigestive germ cell tumors are almost exclusively teratomas, rarely yolk sac tumors.18

Identification of clear cut epithelial differentiation rules out many of the tumors in one's differential for undifferentiated malignancy (although one should remember that some mesenchymal tumors can rarely show immunoreactivity with antibodies to epithelial antigens, eg, olfactory neuroblastomas and primitive neuroectodermal tumor).19,20 For tumors without obvious differentiation seen histologically, immunohistochemistry can be used. Epithelial differentiation can almost always be demonstrated for NMCs with a pankeratin immunococktail. Negative results for other antigens such as S100 protein, CD45, muscle antigens, neuroendocrine antigens, and germ cell antigens are also helpful. p63 immunoreactivity is also helpful as it points toward squamous differentiation and was seen in 4 of 5 NMCs that we tested. It should be remembered, however, that p63 also is expressed by salivary gland tumors that have myoepithelial differentiation such as myoepithelial carcinomas and adenoid cystic carcinomas, and that these tumors can seem undifferentiated, especially on small biopsy.21,22 Using antibodies to other myoepithelial antigens, such as smooth muscle actin, may be helpful to exclude some salivary gland-type carcinomas. Reactivity with antibody to p63 may make some diagnoses, such as sinonasal undifferentiated carcinomas or pituitary adenoma, less likely (Table 3).23

NMCs are not associated with carcinoma-related viruses such as EBV and HPV.10 In situ hybridization for these viruses can be used and the identification of the viruses likely excludes the diagnosis of NMC. Surrogate markers such as p16 immunohistochemistry for the diagnosis of HPV-related malignancy have not been studied.

The definitive diagnosis of NMC and the distinction of such a tumor solely on the basis of histology and immunohistochemistry is not considered possible at this point (see above). Nevertheless, the emergence of a new, very specific monoclonal antibody to NUT holds promise, and may, after further study, prove to be diagnostic. The importance of the accurate diagnosis of NMC is underscored by its prognostic value, and by a move toward more individualized therapy for this disease (unpublished observations).

TREATMENT AND PROGNOSIS

There is no established treatment for NMCs. Most patients have received combination multidrug chemotherapy and radiation. Only occasional cases have undergone subsequent resection. Of the patients with adequate follow-up, all but 1 (15/16) have died of disease with an average survival time of 9 to 10 months.10 It is interesting to note that the single patient with an NMC who survived for an extended period of time had had a tumor of the iliac bone that had originally been diagnosed as Ewing sarcoma and had been treated as such.15 As a result, several patients have been treated with this regimen (Euro Ewing 99), though unsuccessfully (unpublished observations).

CONCLUSIONS

NMCs are uncommon, very aggressive tumors that primarily develop in the thorax or upper aerodigestive tract of young people, although they can develop in patients of any age. Histologically, the tumors range from entirely undifferentiated malignancies to more obvious squamous cell carcinomas. Almost all tumors are immunoreactive with antibodies to keratins and many are immunoreactive with antibodies to p63. As the histology and immunohistochemistry of these tumors (excluding using antibody for the NUT protein) are currently nonspecific, a definitive diagnosis requires molecular testing, preferably FISH, to demonstrate a break of the NUT gene.